A long-term goal of our research is to understand the molecular mechanisms underlying the initiation of cellular DNA replication. Initiation is a defining commitment to cell proliferation; inappropriate onset of replication can lead to genetic instabilities, DNA damage, and changes in gene copy number. From a biomedical perspective, initiation is a keystone pathway that should be susceptible to therapeutic intervention for controlling bacterial infections and cancers; however, a molecular-level understanding of initiation factors and activities is insufficiently complete to advance such efforts. The present application focuses on the initiation of DNA replication in bacteria, using E. coli as a model system. Although a basic framework for this process has been in place for more than 25 years, its mechanistic principles have remained highly enigmatic. By employing an innovative mix of structural methods, new biochemical assays, and analytic technologies, we will answer fundamental questions involving how initiation proteins collaborate to open a bubble in a replication origin and deposit ring-shaped helicases onto the DNA. We will determine in molecular detail: 1) how the DnaA processes the replication origin, oriC, prior to loading of the DnaB helicase and how the ATPase activity of DnaA controls this activity, 2) how the replicative helicase loader, DnaC, coordinates ATP turnover and ssDNA binding to efficiently load DnaB onto DNA and promote helicase-mediated DNA unwinding, and 3) how partner proteins of DnaB coordinate the transition from helicase recruitment to helicase loading, and how they regulate different DnaB translocation activities. The outcome of the proposed studies will be a structural and functional picture of the major steps involved in converting a duplex chromosomal region into a bidirectional replication fork. These findings in turn will: 1) define new principles for both the field of DNA replication and the broader action of ATP- dependent machines and switches, and 2) establish new reagents and assays for advancing drug-discovery efforts that target initiation systems.